Viral hemorrhagic fevers caused by Ebolavirus (EBOV) and Marburgvirus (MARV), both members of the Filoviridae family, are known to be among the most severe infectious diseases in human and nonhuman primates (NHPs), and no licensed vaccines or effective therapeutics are currently available. Zaire ebolavirus (ZEBOV), in particular, has been responsible for multiple Ebola hemorrhagic fever (EHF) outbreaks with case-fatality rates ranging from 65 to 90%. Studies with animal models and limited clinical data from EHF outbreaks suggest that interdependent pathogenic processes, including both the host immune and pathophysiological responses, induced by EBOV infection trigger severe hemorrhagic syndrome. However, in order to develop effective treatments for EHF, it is necessary to better understand the mechanisms of viral and host interactions at the molecular and cellular levels and how these interactions contribute to the in vivo pathogenic process. Therefore, our research is focused on elucidating the roles of viral proteins in the viral replication cycle and pathogenesis. To accomplish this, we have three ongoing projects: (1) the development of a small animal model that recapitulates EHF, (2) the development of efficient reverse genetics systems for generating recombinant ZEBOV from cDNA, and (3) the development of molecular tools for identifying host cellular factors essential for ZEBOV replication. (1) The development of a small animal model that recapitulates EHF. While the NHP model is used to evaluate the efficacy of EBOV vaccines and therapeutics because it accurately recapitulates disease, rodent models (mice and guinea pigs) are convenient and suitable for elucidating the roles of specific viral proteins in the pathogenic process and have been widely used in numerous aspects of EBOV research. However, rodent models produce only limited and inconsistent coagulation abnormalities, which are a hallmark clinical feature of EHF. Therefore, we attempted to develop an EHF rodent model that displays severe coagulation impairment, specifically one that presents with disseminated intravascular coagulation (DIC), a key aspect of EBOV pathology in humans and NHPs. Initially, we demonstrated that rhesus macaques infected with ZEBOV showed severe coagulopathy, as indicated by prolonged coagulation times and decreased fibrinogen and protein C levels. Next, we examined the virulence of mouse-adapted Zaire Ebolavirus (MA-ZEBOV) in Syrian golden hamsters, since MA-ZEBOV, unlike wild-type ZEBOV, produces lethal illness in mice and guinea pigs. Hamsters infected with MA-ZEBOV succumbed to infection and displayed coagulation abnormalities as well as other clinical features that resembled EHF seen in rhesus macaques and humans. The hamster will represent an innovative and powerful rodent model for the evaluation of EHF prophylactic and post-exposure interventions prior to testing in NHPs. Detailed characterization of ZEBOV pathogenesis in this hamster model is ongoing. (2) The development of efficient reverse genetics systems for generating recombinant ZEBOV from cDNA. We have been developing a more efficient rescue system to generate recombinant ZEBOV. We have developed full-length ZEBOV genome clones for wild-type and mouse-adapted ZEBOV. These clones will be used to elucidate the functions of viral proteins and their roles in the viral replication cycle and pathogenesis. (3) The development of molecular tools for identifying host cellular factors essential for ZEBOV replication. We have established various ZEBOV viral protein mutants possessing tandem-tags and fluorescent protein-tags to examine protein-protein interactions among viral and cellular proteins and to identify those cellular interaction partners that are essential for the ZEBOV replication cycle. We have made significant progress in understanding the molecular basis underlying ZEBOV pathogenesis, and we have established or initiated several additional research systems to study host-ZEBOV interaction. We have already developed a small animal model for accurately recapitulating EHF, and we are currently developing a more efficient reverse genetics system as well as various molecular tools for studying ZEBOV. Our work has produced, and will continue to produce, powerful tools for dissecting filovirus pathogenesis and revealing the molecular details behind host-filovirus interactions.